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Cell-specific role of Hfe in Salmonella infection
groups existed. For the comparison of organ bacterial loads and mRNA expression, data were log-transformed prior to Student’s t-test. ANOVA with Bonferroni correc- tion was used when more than two groups existed. Survival was compared by log-rank test. Generally, P-values less than 0.05 were considered significant.
Results
Hepatocyte-specific Hfe deletion stimulates extracellular growth of Salmonella Typhimurium
We previously reported that mice lacking Hfe in all cell types (Hfe-/- mice) were partially protected from S. Tm. Infection.13,20
Consistently, we could recapitulate this finding in a dif- ferent strain of Hfe-/- mice in which exons 3-524 rather than exons 2-313 of Hfe were deleted. We found that also these Hfe-/- mice survived significantly longer (Figure 1A) and carried reduced numbers of bacteria in spleen, liver and serum in response to S. Tm infection when compared to Hfe+/+ littermates (Figure 1B to D). In order to delineate in which cell type the absence of Hfe confers protection from infection, we next analyzed mice with selective Hfe-deficiency in hepatocytes (referred to as AlfpCre+ Hfefl/fl). Previous analyses of the AlfpCre+ Hfefl/fl line showed an iron phenotype comparable to Hfe-/- mice,25 with elevated iron levels in serum and liver and iron defi- ciency in the spleen.
AlfpCre+ Hfefl/fl and control mice (AlfpCre- Hfefl/fl) were infected with S. Tm. and survival time was monitored for 14 days (336 hours). Unexpectedly and in contrast to the previous findings in Hfe-/- mice, we observed significantly shortened survival in the AlfpCre+ Hfe-/- mice (Figure 1E). Bacterial burden in spleen and liver was not substantially altered, when compared to control mice (Figure 1F and G). By contrast, the number of bacteria circulating in the serum was significantly higher in AlfpCre+ Hfefl/fl mice (Figure 1H). This finding suggested that Hfe-deficiency in hepatocytes does not confer protection against S. Tm. infection related death but even aggravates the infection phenotype.
Macrophage-specific Hfe-deletion phenocopies the protective effect of constitutive Hfe deletion in mice infected with Salmonella Typhimurium
We next tested the response to S. Tm. infection in mice lacking Hfe in myeloid cells (referred to as LysMCre+ Hfefl/fl)25 in comparison to control mice (LysMCre– Hfefl/fl). Interestingly, macrophage-specific Hfe depletion fully recapitulated the protective effect observed in Hfe-/- mice, including prolonged survival (Figure 1I) and reduced bac- terial load in spleen, liver and serum (Figure 1J to L). Importantly, the alleles required for tissue-specific recom- bination to generate the cell type-specific Hfe-depletion models, LysMCre (macrophage-specific Cre-recombinase expression) and AlfpCre (hepatocyte-specific Cre-recombi- nase expression) alone had no effect on survival and bac- terial burden in the spleen, liver and serum (Online Supplementary Figure S1A to D and 26), excluding non-spe- cific effects of the Cre-recombinases. We conclude that the lack of Hfe in myeloid cells is sufficient to protect mice from S. Tm. infection related consequences. This finding demonstrates an important extra-hepatic function of Hfe in vivo.
Salmonella-infection of LysMCre+ Hfefl/fl mice causes iron depletion in macrophages
In order to understand the mechanism underlying diver- gent disease outcomes of S. Tm. infection in AlfpCre+ Hfefl/fl and LysMCre+ Hfefl/fl mice, we analyzed iron-related parameters. Iron localization was detected in tissue sec- tions of Salmonella-infected mice by Prussian blue staining and tissue iron levels were quantified by colorimetric measurement. S. Tm.-infected Hfe-/- (Figure 2A and B) and LysMCre+ Hfefl/fl mice (Figure 2G and H) showed reduced iron levels in the spleen consistent with the protective phenotype observed in these mouse strains. This was not apparent in infected AlfpCre+ Hfefl/fl mice (Figure 2D and E). By contrast, infected Hfe-/- (Online Supplementary Figure S2A and B) and AlfpCre+ Hfefl/fl mice (Online Supplementary Figure S2C and D) showed hepatocellular iron accumula- tion, while liver iron levels were normal in infected LysMCre+ Hfefl/fl mice (Online Supplementary Figure S2E and F). Importantly, the reduction of splenic iron levels in infected Hfe-/- and LysMCre+ Hfefl/fl mice correlated with diminished intracellular iron levels in bone marrow macrophages (Figure 2C and I), while AlfpCre+ Hfefl/fl bone marrow macrophages had a normal iron content (Figure 2F). This finding suggests that upon Salmonella infection, macrophages lacking Hfe show reduced iron levels.
High serum iron in AlfpCre+ Hfefl/fl mice allows for increased proliferation of Salmonella
Hfe-/- and AlfpCre+ Hfefl/fl mice infected with S. Tm. WT for 72 hours showed elevated serum iron levels compared to Hfe+/+ or AlfpCre– Hfefl/fl mice, respectively (Figure 3A and B). In contrast, serum iron levels in infected LysMCre+ Hfefl/fl mice were comparable to infected LysMCre– Hfefl/fl mice (Figure 3C). Notably, in the setting of Salmonella infection, serum levels of hepcidin-1 were not different between the mouse strains (Figure 3D to F). Moreover, Salmonella-infected Hfe-/- mice presented with increased serum concentrations of the siderophore-capturing pep- tide Lcn2 (Figure 3G) while hepatocyte-specific (Figure 3H) or macrophage-specific (Figure 3I) Hfe deletion did not affect serum Lcn2 levels. Thus, the presence of Hfe in hepatocytes is necessary and sufficient to limit serum iron levels both in steady state25 and in response to S. Tm. infection. Moreover, hepcidin-1 induction in response to Salmonella infection is appropriate in mice lacking Hfe. In contrast, the enhanced production of Lcn2 is only observed in the complete absence of Hfe13 suggesting that different cell-types mediate iron- and immune-regulatory effects of Hfe.
Salmonella iron acquisition pathways differently affect extracellular proliferation
S. Tm. is a bacterial pathogen with dual lifestyle. First, S. Tm. is able to persist and replicate extracellularly, e.g., on contaminated food and surfaces, in the gut lumen and in the serum. Early after the invasion of a murine host, S. Tm. preferentially infects macrophages to propagate intracellularly. We therefore investigated how serum iron availability in Hfe-/-, AlfpCre+ Hfefl/fl and LysMCre+ Hfefl/fl mice may affect bacterial proliferation. We spiked RPMI medium with 10% of serum from uninfected mice of all three strains and inoculated spiked samples with bacteria. We used S. Tm. WT and isogenic mutants lacking either single or all three major bacterial iron uptake systems (enterobactin, feo and sitABCD).27 In addition, we includ-
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